Methods to determine cytokine protein content in samples of interest, such as enzyme-linked immunosorbent assay (ELISA), are often labor-intensive and costly. Furthermore, because ELISA requires relatively large sample volumes and protein concentrations, it is difficult using this technique to determine protein content for multiple cytokines from individual samples. Recently, Luminex has developed an open source hardware platform combining flow cytometry- and bead-based antibody capture that is capable of detecting multiple analytes from a single sample. In the present study we employed the Luminex 200 platform to determine the cytokine protein content in discrete brain regions of C57BL/6J mice. In spike-and-recovery experiments, known concentrations of murine recombinant interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)alpha were added either singly or as a mixture of all three to whole brain homogenates containing known quantities of total protein. Spiked samples were assayed for either a single cytokine or for multiple cytokines using 1-plex or 3-plex assay kits, respectively. In whole mouse brain homogenate we recovered between 81% and 103% of the recombinant cytokines. We then injected C57BL/6J mice intraperitoneally with bacterial lipopolysaccharide (LPS) and sacrificed them 4h later. We detected in samples taken from LPS-stimulated mice 4- to 870-fold increases in serum or spleen cytokine protein, and 1.5- to 16-fold increases in cytokine protein in discrete brain regions, relative to protein content in samples obtained from vehicle-treated animals. These results indicate that multiple cytokines may be reliably assayed from discrete regions of mouse brain using a single sample.

译文

确定目标样品中细胞因子蛋白含量的方法,例如酶联免疫吸附测定 (ELISA),通常是劳动密集型且昂贵的。此外,由于ELISA需要相对较大的样品体积和蛋白质浓度,因此使用该技术很难确定来自单个样品的多种细胞因子的蛋白质含量。最近,Luminex开发了一种结合流式细胞术和基于珠子的抗体捕获的开源硬件平台,该平台能够从单个样品中检测多种分析物。在本研究中,我们使用Luminex 200平台来确定C57BL/6J小鼠离散脑区域中的细胞因子蛋白含量。在峰值和恢复实验中,已知浓度的鼠重组白细胞介素 (IL)-1β,IL-6,并且将肿瘤坏死因子 (TNF) α 单独或作为所有三种混合物添加到包含已知量的总蛋白的全脑匀浆中。使用1-plex或3-plex检测试剂盒对加标样品进行单一细胞因子或多种细胞因子的测定,分别。在整个小鼠脑匀浆中,我们回收了81% 至103% 的重组细胞因子。然后,我们向C57BL/6J小鼠腹腔注射细菌脂多糖 (LPS),并在4小时后将其处死。我们在从LPS刺激的小鼠身上采集的样本中检测到血清或脾脏增加4至870倍细胞因子蛋白,相对于从媒介物处理的动物获得的样品中的蛋白质含量,离散脑区域中的细胞因子蛋白增加1.5至16倍这些结果表明,使用单个样品可以从小鼠脑的离散区域可靠地测定多种细胞因子。

+1
+2
100研值 100研值 ¥99课程
检索文献一次
下载文献一次

去下载>

成功解锁2个技能,为你点赞

《SCI写作十大必备语法》
解决你的SCI语法难题!

技能熟练度+1

视频课《玩转文献检索》
让你成为检索达人!

恭喜完成新手挑战

手机微信扫一扫,添加好友领取

免费领《Endnote文献管理工具+教程》

微信扫码, 免费领取

手机登录

获取验证码
登录